Phagocytes such as macrophages and neutrophils provide a primary line of defense against a variety of diseases, including those caused by infectious agents and cancers (Gomme and Bertolini, 2004). During a study of the role of inflammation in development of immunity, Yamamoto and Homma (1991) discovered that a serum protein was required to activate macrophages. This protein is the vitamin D-binding protein (DBP). The human protein is known as group-specific component, or Gc protein. DBP is an abundant, multifunctional, polymorphic glycoprotein in human serum. Highly conserved homologs of this protein occur among all mammalian species (Yang et al., 1990; White and Cooke, 2000). As its name implies, one role of the protein is as a vehicle for circulating vitamin D in blood. Another function involves binding of actin released into the blood during tissue injury. The glycan of the serum protein can be processed to a potent anti-cancer agent, which is expressed through its macrophage activation and anti-angiogenesis activities (Kanda et al., 2002; Gomme and Bertolini, 2004).
DBP is a 458-amino acid protein in humans and consists of three major domains similar to albumin (Head et al., 2002; Otterbein et al., 2002; Verboven et al., 2002). DBP is a glycoprotein that carries a single trisaccharide group (Yang et al., 1985; Cooke and David, 1985). The O-linked glycan is found in the carboxy-terminal Domain III, attached to the hydroxyl group of a specific threonine residue (Thr420 in protein from human). Its structure has been determined as NeuNAc(α2→3) Gal(β1→3) GalNAc(α1→O) Thr, with significant amounts of the O-glycan found only on the Gc1 isoform (Coppenhaver et al., 1983; Viau et al., 1983). Some of the glycans contain a second NeuNAc linked α2→6 to GalNAc. Extensive work by Yamamoto and colleagues (Yamamoto and Kumashiro, 1993; Yamamoto and Naraparaju, 1996 a,b) suggested that DBP has remarkable therapeutic value as an activator of macrophages. Its potent stimulatory activity for macrophage phagocytosis is expressed when its glycosylated site is processed to a single O-linked GalNAc by removal of the NeuNAc (sialic acid) and the Gal residues (Yamamoto and Homma, 1991; Yamamoto and Kumashiro, 1993). The precursor protein can be processed to the active form in vitro by treatment with immobilized sialidase and β-galactosidase (Yamamoto and Kumashiro, 1993; Yamamoto and Naraparaju, 1998). In animals, the modified protein is referred to as DBP-MAF, whereas the active form of the human protein is known as Gc-MAF. These designations are used interchangeably. The active form of the protein reduces tumor cell load (Kisker et al., 2003; Onizuka et al., 2004), provides a therapy against viral infections such as HIV (Yamamoto et al., 1995), and promotes bone growth (Schneider et al., 1995; 2003) and therapy against bone disorders such as ostepetrosis (Yamamoto et al., 1996b). DBP-MAF has also been found to be an effective anti-angiogenesis factor (Kanda et al., 2002; Kisker et al., 2003) and is a potent adjuvant for immunizations (Yamamoto and Naraparaju, 1998). A lectin receptor that specifically binds GalNAc residues was identified on the surface of human macrophages (Iida et al., 1999).
Cancer cells secrete, and some virus particles carry on their surface, an enzymatic activity (N-acetylgalactosaminidase) that depletes the precursor protein in the serum by removing the O-glycoside, which renders the protein inactive as a macrophage activating factor (Yamamoto et al., 1996a, 1997). A decrease in active Gc-MAF may be a major factor in progression of disease. Introduction of the in vitro processed protein leads to dramatic reduction in the amount of cancer cells in animals (Yamamoto and Naraparaju, 1997; Kanda et al., 2002; Kisker et al., 2003; Onizuka et al., 2004) and appears to also reduce the number of HIV particles in infected individuals (Yamamoto et al., 1995). This conclusion is based largely on the decrease in activity of N-acetylgalactosaminidase, whose level appears to be directly correlated with tumor and viral loads in cancer and in HIV-infected patients, respectively (Yamamoto et al., 1997).